Unilamellar phospholipid micellular particles in the form of vesicles (also known as liposomes) have received increasing attention from researchers as carriers of various substances, such as imaging agents and for diagnosis of abnormalities such as tumors in humans using animal models. In particular, it has been shown that small vesicles (less than 2000 .ANG.) may be labelled to target tumors (Proffitt, et al., J. Nucl. Med. 24(1), p. 45-50 (1983)) incorporated hereinafter by reference. Such vesicles are also useful as potential carriers of therapeutic agents for treatment of tumors. Alternatively, small vesicles are useful for in vitro immunoassays. U.S. Pat. No. 4,342,826 and D. Papahadjopoulos (Ed.) Annals N.Y. Acad. Sci., 308 (1978). Additionally, the vesicles containing imaging or therapeutic agents may be modified by incorporating various carbohydrate derivatives into the vesicle surface to increase tissue specificity of the vesicles, or by adding cholesterol to increase the stability of the vesicles. Mauk and Gamble, Anal. Bioc. 94, pg. 302-307 (1979); Mauk, et al., P.N.A.S. (U.S.A.) 77(8), pg. 4430-4434 (1980); and Liposome Technology, Targeted Drug Delivery and Biological Interaction, Vol. III, G. Gregoriadis (Ed.), C.R.C. Press, Inc. (1984), all of which are incorporated herein by reference.
The prior art shows that vesicles such as liposomes may be produced using the methods of sonication, dialysis, injection or reverse phase evaporation. These procedures are well known and may be found in the following articles: Huang, Biochemistry 8, pg. 344 (1969) (Sonication); Rhoden and Goldin, Biochemistry 18, pg. 4173 (1979) (dialysis); and Kremer et al Biochemistry 16, pg. 3932-3935 (1977) (injection); and Liposome Technology, Preparation of Liposomes, Vol. I, 6 Gregoriadis (Ed.), CRC Press Inc. (1984), all of which are incorporated herein by reference. These methods share several disadvantages including the inability to conveniently produce commercial quantities of such vesicles.
The use of homogenizing devices to produce emulsions from solutions with soluble and insoluble components is well known in the art. U.S. Pat. No. 4,127,332. Several such homogenizing devices operate by creating shearing forces to disperse the insoluble and soluble components. These shearing forces result from the process known as cavitation which involves the rapid formation of bubbles within the sample solution as it passes through narrow channels causing a reduction in the vapor pressure of the fluid. The bubbles then collapse as the solution moves out from these channel areas, generating a shearing force. Such homogenizing devices, however, have been operated at relatively low pressures (usually below 10,000 psi) for the purpose of creating emulsions with large particles (greater than 1 micron) such as lipoproteins for baking purposes, (U.S. Pat. No. 4,360,537), or simply to form an emulsion of oil and water. U.S. Pat. No. 4,026,817.
Recently, various mechanical devices such as homogenizers have been employed in producing vesicles. U.S. Pat. No. 4,411,894. However, these devices have been used to assist with the initial dispersion of vesicle precursor substances such as soya or egg lecithin which do not require high shear forces to form vesicles and which do not form vesicles optimally stable in vivo. In addition, the French Press and Pressure Cell has been used to generate small vesicles. U.S. Pat. No. 4,263,428. A disadvantage of this device is that it requires extra time to reload a sample since it provides no means to recirculate the lipid solution through the device.
It is, therefore, an object of the present invention to provide an efficient, time-saving and reproducible process, having the advantages enumerated above for producing commercial quantities of small, unilamellar vesicles, especially vesicles suitable for treatment and diagnosis of tumors in a body.